The overall goal of this proposal is to develop a more detailed understanding of the molecular basis of biological electron transfer. Electron transfer is of central importance in all biological redox reactions and in energy conversion in the mitochondria and chloroplast, but little is known about the role that protein structure and protein and cofactor conformational changes play in this process. The spectroscopic studies to be described here will address these questions; the model system that will be used is the photosynthetic reaction center. The factors that influence the efficiency and directionality of electron transfer after light absorption in the reaction center are likely to be important in other redox reactions. Moreover, photosynthetic reaction centers provide a unique system for study of electron transfer mechanism, because the reactions can be initiated by light. The specific aims of this proposal are to use electron paramagnetic resonance (EPR) and difference Fourier- transform infrared (FT-IR) spectroscopy to study light-driven electron transfer events in bacterial reaction centers and in photosystem II, the water-splitting complex. EPR experiments on photosystem II will be aimed at the identification of an intermediate electron transfer component, Z+, of photosystem II, which is light-induced and is believed to be an amino acid residue. Collaborative experiments will explore the spectroscopic properties of mutants where other amino acids have been substituted at the putative Z+ site. This work will define the structural features that are important in electron transfer on the donor side of photosystem II. Difference FT-IR experiments will be performed on both the bacterial reaction center and on photosystem II. Subtraction of a "dark" from a "light" spectrum makes this technique specific for vibrational changes associated with charge separation. In the bacterial reaction center, difference FT-IR spectra will contain information about the structural changes in protein residues and in cofactors that accompany charge separation. In photosystem II, difference FT-IR will provide a new technique to use in studies of conformational changes in the manganese cluster, the site of water oxidation, and in the study of the protein structural alterations, such as protonation/deprotonation events, that are involved in the formation of Z+. The key to interpretation of both EPR and FT-IR spectra is isotopic labeling through the use of auxotrophs; bacteriochlorophy11, chlorophy11, quinones, and protein amino acid residues can all be labeled in vivo.